The present invention relates to a composition and a drug, wherein flavonoids in the licorice are used for the antivirus agents, in particular, for the preventive therapy against AIDS [Acquired Immunodeficiency Syndrome] virus.
AIDS has become now a worldwide problem as a disease wherein said virus carriers amount presumably four million (end of the year 1987) and the mortality within two years after onset exceeds 70%. A feature of AIDS is that the T4 lymphocytes governing the immune system are destructed by AIDS virus resulting in the collapse of both of bacillary immunity and liquid immunity and finally the death comes from Karini's pneumonia, Kaposi's sarcoma, etc.
At the research organs in U.S.A. and other countries, the development of therapeutical drugs and onset-preventive drugs for AIDS is in haste, but, until today, only one kind of azidothymidine being of nucleic acid type is approved as a therapeutical drug for AIDS. However, even with said azidothymidine, the side-effects such as severe anemia, granulocytopenia, etc. are strong leaving a problem in the administration for a long term.
With glycyrrhizin being an effective ingredient of herbal medicine "licorice", the side-effect is hardly recognized. Thus, it is attracting the attention as an anti-AIDS agent permitting the long-term administration, but the anti-AIDS virus activity thereof is low and it has not been put into practice.
While, until today, any anti-AIDS virus active ingredient other than glycyrrhizin has not been found in the licorice, but, in said herbal medicine, triterpene glycosides such as glavaric acid etc., flavonoids such as liquiritin, i-liquiritin, liquiritigenin, i-liquiritigenin, glycyrol, licoricidin, etc. and physiologically active substances such as .beta.-sitosterol, biotin, etc. are contained and the possibility of these ingredients to exhibit the anti-AIDS virus activity is also left behind. There, on the basis of knowledges and ideas aforementioned, whether or not there are substances which exhibit the antivirus activity (Newcastle Disease Virus, hereinafter referred to as NDV) except glycyrrhizin among the ingredients of licorice was first tested.
First, the tests for the anti-NDV activity of glycyrrhizin (content of glycyrrhizin: not less than 98%, hereinafter referred to as glycyrrhizin standard) and refined product of extract from licorice with aqueous ammonia (content of glycyrrhizin: 40%, hereinafter referred to as BS) was carried out in vitro. As a result, the anti-NDV activity was recognized for both, but BS showed the activity about ten times stronger and it became evident that there existed the antivirus active substances other than glycyrrhizin in the licorice.
Now, in order to clear up the substantiality of anti-NDV activity shown with BS, BS was fractionated in sequence by means of medium-pressure liquid chromatography and high-performance liquid chromatography to obtain a fraction (hereinafter referred to as BS-F) showing the activity scores times as strong as that of BS. This BS-F is yellow needle-like crystals and easily soluble into alcohol and ether and hardly soluble into water.
From these properties, BS-F was presumed to be a kind of flavonoids, and the identification test appearing in Japan Pharmacopeia, that is, the color identification test wherein BS-F is dissolved into ethanol and hydrochloric acid and magnesium are added was conducted. As a result, purplish red color was displayed showing the peculiar reaction of flavonoids.
Further, silica gel thin-layer chromatography (development solvent; acetone: benzene: i-amyl alcohol: water: 28% aqueous ammonia (10:3:3:1.5:0.5), coloring agent; acetic anhydride: sulfuric acid (3:1)) according to the method described in Japanese Patent Publication No. Sho 48-8458 and .sup.13 C-nuclear magnetic resonance spectrography were performed to identify BS-F being liquiritigenin. Besides, it should be noted supplementarilly that, also in infrared absorption spectrum, partial absorptions of flavonoid skeleton at 1660 cm.sup.-1 (&gt;C.dbd.O) and 1240 cm.sup.-1 (C--O--C) were seen.
Based on the knowledges above, the anti-NDV activity tests were conducted similarly with licorice flavonoids other than liquiritigenin (liquiritin, i-liquiritin and i-liquiritigenin). At that time, all of the flavonoids were confirmed to have the anti-NDV activity, though weaker than that of liquiritigenin.
Further, the antivirus activity tests were performed replacing virus with Rous Sarcoma Virus (hereinafter referred to as RSV), which belongs to the same retrovirus family as AIDS virus does. As a result, the anti-RSV activity was seen with every flavonoid. In particular, liquiritigenin was confirmed to have strong activity. Thus, test against the AIDS virus, which is the final purpose of the present research, was conducted.
Consequently, all of the flavonoids inhibited the proliferation of AIDS virus. And yet, the proliferation-inhibitory effects shown therewith were made clear to be considerably stronger than that of glycyrrhizin, leading to the completion of the invention.